Table 1.
Bifidobacterium longum general genome features.
Fig 1.
Phylogenetic tree based on the B. longum core-genome.
(A) The B. longum subsp. longum phylogenetic group. (B) The B. longum subsp. infantis phylogenetic group. B. longum subsp. longum and B. longum subsp. infantis type strains are indicated in blue text. Lactobacillus salivarius was included as an outlier.
Fig 2.
Illustration of the EPS cluster located in the B. longum 35624 genome and comparison to similar clusters located in B. longum 105-A, B. longum subsp. longum JCM1217 and B. longum subsp. longum NCC2705. Each gene is colour-coded according to function which is indicated in the legend located at the end of the page. Percentages represent the percent of sequence similarity at the protein level with corresponding genes in the B. longum 35624 genome. The locus tags of the first and last genes located in the EPS clusters of B. longum 105-A, B. longum subsp. longum JCM1217 and B. longum subsp. longum NCC2705 are also indicated in the illustration.
Fig 3.
B. longum 35624 electron microscopy.
(A) A layer of extracellular polysaccharide is clearly visible by electronic microscopy of the 35624 strain. (B) The isolated and purified EPS is illustrated.
Fig 4.
B. longum 35624 EPS characterization.
(A) Comparison of the EPS (solid line) with a dextran standards dotted line) demonstrated that EPS had an average molecular mass much higher than 1 MDa (Mw). (B) HPLC analysis of anthranilic acid-labeled monosaccharides of EPS revealed the presence of glucose (Glc), galactose (Gal), some galacturonic acid (GalA) and two additional peaks with masses corresponding to an aldobiuronic acid and a deoxy-hexose later identified as 6-deoxy-talose. The upper trace in (B) is the standard mixture and the lower trace shows the results of the EPS sample. Numbers in the lower trace give the masses of the compounds as determined by off-line ESI-MS.
Fig 5.
(A) Separation of EPS fragments by PGC HPLC with MS/MS detection. The extracted ion chromatogram for mass 1008.39 Da shows four peaks. Their reducing end sugar was clearly revealed by ESI-MS/MS. Their assignment as either Gal or Glc and the interpretation in terms of fragment structures was done a posteriori based on MALDI-TOF data and on knowledge of the EPS structure. (B) Example of a MALDI-TOF/TOF fragment spectrum showing b-ions from the non-reducing and y- and y´ (= 1,5x) -ions from the reducing end.
Fig 6.
B. longum 35624 EPS characterization.
(A) The 600 MHz 1H NMR proton spectrum of the acid-treated 35624 EPS (D2O, 338 K) is illustrated. A part of the high-field region is displayed in the insert. (B) Expansion plot of the 150 MHz 13C NMR spectrum of the acid-treated 35624 exopolysaccharide. The anomeric signals on the left confirmed the presence of a hexasaccharide repeat unit.
Fig 7.
B. longum 35624 EPS proton and carbon signals.
(A) A selected region of the multiplicity-edited, gradient enhanced 1H, 13C-HSQC NMR spectrum of the exopolysaccharide. Letters denote the residues as given in the structural formula and arabic numerals denote the respective pyranose position. Resonances from anomeric carbons/protons, glycosylation sites and resolved signals are annotated. (B) Selected region of the 1H, 13C-HSQC-TOCSY NMR spectrum (600 MHz) of the acid-treated 35624 EPS. Arabic numerals before and after oblique stroke denote carbons and protons, respectively.
Table 2.
1H and 13C NMR chemical shifts (δ, ppm) of the exopolysaccharide (recorded at 338 K) and the tetrasaccharide os211 (recorded at 300 K) from B. longum 35624.
Fig 8.
B. longum 35624 EPS composition and structure.
The structure is annotated as the chemical formula and in condensed form. Capital letters denote the residues as in Figs 6 and 7.